GSM7474209: control, replicate 4 [NS41]; Caenorhabditis elegans; RNA-Seq
For isolation of the GLR glia, synchronized L1 larvae expressing nuclear localized YFP in the GLR glia cells (OS11715) were grown on thirty 10cm plates seeded with HB101 E. coli bacteria. After 48 hours at 20oC, L4 larvae animals were washed off plates and subsequently washed ten times with M9 to remove excess bacteria. Each wash consisted of a brief (10 second, 1300 rpm) centrifugation, such that most animals were pelleted, but bacteria stayed in suspension. Animals were then dissociated using SDS-DTT (0.25% SDS; 200 mM DTT; 20 mM HEPES, pH 8.0; 3% sucrose) and Pronase E (15 mg/ml). We used 2:1 ratio of SDS-DTT to a volume of packed animals pellet, followed by 4 min incubation on ice. After washes, 4:1 ratio of Pronase E was added to the packed animals pellet and animals were incubated rotating at 20 °C for 5 min, followed by 12 min of gentle homogenization (7 mL dounce homogenizer, pestle clearance 0.02−0.056 mm, Kimble Chase). After washes with ice-cold egg buffer (1.18 M NaCl; 480 mM KCl; 20 mM CaCl2; 20 mM MgCl2; 250 mM HEPES, pH 7.3) to remove Pronase E, cells were filtered through a 5μM filter to remove undigested animal fragments, and immediately sorted by FACS. mRNA amplification and cDNA preparation were performed using the SMARTer mRNA amplification kit (Takara #634940) according to manufacturer's instructions.